(D-124) Drug Delivery Application of Redox-Responsive and Dual-Targeted Folate-Conjugated Hyaluronic Acid-s-s-Gold nanoparticles For the Cancer Therapy

Introduction:: Methotrexate (MTX) has anti-cancer drug effects on breast, gastric, prostate, lung cancers, head, neck, and esophagogastric carcinomas. However, its potential toxicity and limited therapeutic efficacy are significant challenges. Targeted drug delivery systems, particularly nanotechnology-based folate platforms, have been proposed to address these issues. In this study, we developed folic acid (FA)-conjugated hyaluronate (HA)-s-s-Gold nanoparticles (FA-HA-s-s-Gold NPs) as a targeted and controlled drug delivery system for cancer treatment.

Gold NPs thiolated and coated with MPA (3-mercaptopropionic acid) have several advantages in photothermal therapy and drug delivery systems due to their small particle sizes and inertness. Hyaluronic acid has a strong affinity for cell-specific surface markers such as Cluster of Differentiation 44 (CD44) and Receptor for Hyaluronic Acid-Mediated Motility (RHAMM) receptors, which are abundantly overexpressed on the surface of many types of tumors. HA, and its derivatives are widely used as ligands for tumor-targeted drug delivery. Dual receptor targeting, a novel strategy for targeted drug delivery, can effectively and specifically target nanoparticles to tumor cells. Our study used a straightforward and scalable procedure to create the nanoparticles. The HA polymer was selected as the main chain for hydrophobized modification due to its specific targeting of tumor cells via the CD44 receptor. The further conjugation of disulfide bonds and folic acid receptor (FR) to HA NPs achieved dual active targeting and glutathione (GSH) redox-responsive properties. MTX was successfully encapsulated by HA-s-s-Gold and FA-HA-s-s-Gold NPs. The physicochemical characterization, in vitro cytotoxicity, and cellular uptake of the developed nanoparticles were studied in detail.

Materials and Methods:: Gold NPs were synthesized via the modified Turkevich-Frens method. In brief, 50 mL of aqueous HAuCL4.3H2O (0.5 mM) was heated to 100 °C, and aqueous trisodium citrate (40 mM) preheated to 70 °C was added. The mixture was stirred under reflux for 1 hour, cooled to room temperature, and stored at 4 °C.

Gold@MPA NPs were synthesized by adding 0.01 M MPA to the gold colloid solution at pH 11. HA-s-s-Gold NPs were synthesized by first activating the carboxyl group (COOH) of HA (40 mg/mL) using equimolar amounts (2.5 mM) of EDC. HCL and NHS, followed by the addition of cystamine dihydrochloride (CYS) at 0 °C. The formed HA-s-s-NH2 was dialyzed for 2 days and lyophilized. Gold@MPA was activated in the same way and was then chemically grafted to HA-s-s-NH2 (5 mg/mL), stirred for 24 hours and lyophilized. FA-HA-s-s-Gold NPs were synthesized by reacting folic acid (22 mg/mL) with EDC. HCL and NHS at 21 °C overnight in the dark, then the solution was drop-wise added to HA-s-s-Gold NPs for 24 hours. The mixture was first dialyzed against a NaHCO3-Na2CO3 buffer solution (pH 10) for 24 hours, then against DI water for 3 days, and then lyophilized. MTX-loaded NPs were synthesized by dispersing 1 mg/ml of HA-s-s-Gold or FA-HA-s-s-Gold NPs in DI water and then drop-wise adding an aqueous solution of MTX in DMSO. The resulting solution was injected drop-wise (in a G22 syringe) into DI water with gentle stirring overnight, followed by dialysis and centrifugation.


Results, Conclusions, and Discussions:: The chemical structure of formed nanoparticles, including HA, HA-s-s-NH2, FA-HA-s-s-Gold NPs, and DS of CYS on the backbone of HA, was analyzed using 1H Nuclear Magnetic Resonance (NMR) and Fourier Transform Infrared Spectroscopy (FTIR). The 1H NMR spectrum of HA displayed identifiable peaks at 1.95 ppm for N-acetyl, 3.0–4.0 ppm for glucosidic, and 4.35 and 4.45 ppm for anomeric H. Newly emerged peaks at 2.79 ppm indicated the presence of disulfide bonds. Weak signals at 6.6–8.6 ppm, which correspond to the aromatic protons of folic acid, confirmed that folic acid had been grafted onto HA-s-s-Gold NPs. The size of different NPs, such as Gold, Gold@MPA, HA-s-s-Gold NPs, FA-HA-s-s-Gold NPs, and MTX-loaded NPs, varied from 21 nm to 160 nm. Overall, the negative charge zeta potential, UV shift, and FE-TEM studies proved that the nanoparticles were stable. With a high encapsulation efficiency of 98.7%, MTX was successfully integrated into the hydrophobic cores of the NPs. The release profile showed a controlled and sustained release of MTX for 72 hours. In vitro, studies revealed that FA-HA-s-s-Gold dramatically increased the cytotoxicity of HeLa cancer cells when compared to HA-s-s-Gold NPs and free MTX but had no significant impact on HEK-293 cells. Cellular uptake studies conclude that the quantity of FA-HA-s-s-Gold nanoparticles that are internalized in cancer cells was increased.



The HA-s-s-Gold and FA-HA-s-s-Gold amphiphilic nanoparticles developed in this study have the potential to function as biocompatible and biodegradable carriers for targeted medication delivery in the treatment of cancer. They are an appealing platform for future development in cancer therapeutics due to their small particle sizes, high drug encapsulation efficiency and loading capacity, sustained-release properties, and dual targeting capability.



In this study, we synthesized cancer therapeutic nanoparticles. MTX-loaded NPs displayed sustained drug release and increased cytotoxicity against cancer cells, as compared to free MTX. The FA-HA-s-s-Gold NPs additionally demonstrated dual targeting ability against cancer cells expressing CD44 and FR receptors. The HA-Gold system's utilization of gold NPs enables regulated medication release and photothermal therapy. Future in vivo studies are required to assess the efficacy and safety of these NPs for clinical translation.




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